The NFAT (nuclear factor of activated T cells) family comprises several structurally related proteins that are encoded by at least four distinct genes (see e.g., McCaffrey et al. (1993) Science 262:750-754; Northrop et al. (1994) Nature 369:497-502; Masuda et al. (1995) Mol. Cell. Biol. 15:2697-2706; Hoey et al. (1995) Immunity 2:461-472; Ho et al. (1995) J. Biol. Chem. 270:19898-19907). In resting T cells NFAT proteins are present in the cytoplasm as phosphorylated species. Upon activation, sustained increases in calcium activate the phosphatase, calcineurin, which subsequently dephosphorylates NFAT (Beals, C. R., et al. 1997. Genes Dev. 11, 824-834.; Clipstone, N. A. and Crabtree, G. R. 1992. Nature 357, 695-697.; Flanagan, W. M., et al. 1991. Nature 352, 803-807) which is then quickly translocated into the nucleus to drive gene expression in association with other factors such as c-Maf, AP-1 and NIP45 (Cockerill, P. N., et al. 1993. Proc. Natl. Acad. Sci. USA 90, 2466-2470.; Rooney, J. W., et al. 1995. Immunity 2, 545-553.; Ho, I-C., et al. 1996. Cell 85, 973-983.; Hodge, M. R., et al. 1996; Beals, C. R., et al. 1997. Science 275, 1930-1933). The activation-dependent dephosphorylation and translocation of NFAT in lymphocytes and in cardiac endothelial and muscle cells can be blocked by immunosuppressive drugs such as cyclosporin A (CsA) or tacrolimus (FK506) which block calcineurin (Emmel, E. A., et al. 1989. Science 246, 1617-1620).
The cytoplasmic subunit of NFAT is encoded by a family of genes including NFATp, NFATc, NFAT3, and NFAT4/x, all of which bind to and transactivate NFAT target sequences in vitro (see e.g., Hoey et al. (1995) Immunity 2:461-472; Masuda et al. (1995) Mol. Cell. Biol. 15:2697-2706; McCaffrey et al. (1993) Science 262:750-754; Northrop et al. (1994) Nature 369:497-502). These family members share approximately 70% sequence identity within a region related to the Rel homology domain. The first member of the family to be purified and cloned was NFATp (see e.g., McCaffrey et al. (1993) Science 262:750-754). NFATp (also referred to as NFAT1) is constitutively expressed as a cytoplasmic phosphoprotein in resting immune cells (see e.g., Shaw et al. (1995) Proc. Natl. Acad. Sci. 92:11205-11209). Upon stimulation of the immune cells, NFATp is dephosphorylated by a calcium-calcineurin dependent pathway and translocates to the nucleus. Following translocation to the nucleus, nuclear NFAT associates with a nuclear component econtaining fos and jun proteins (see e.g., Rao (1994) Immunology Today. 15:274-281), which are synthesized by stimuli that activate protein kinase C. The associated NFAT complex binds to the IL-2 promoter to initiate expression of the IL-2 gene.
Despite their name, NFAT proteins are not only expressed in T cells, but also in other classes of immune-system cells. NFAT proteins are activated by stimulation of receptors coupled to calcium mobilization, such as the antigen receptors on T and B cells, the Fcs receptors on mast cells and basophils, the Fcxcex3 receptors on macrophages and NK cells, and receptors coupled to certain heterotrimeric G proteins (see e.g., Rao (1994) Immunology Today. 15:274-281; Venkataraman et al. (1994) Immunity 1:189-196; Choi et al. (1994) Immunity 1:179-187; Weiss et al. (1996) Mol. Cell. Biol. 16:228-235; Aramburu et al. (1995) J. Exp. Med. 182:801-810).
Proteins belonging to the NFAT family also play a central role in other inducible gene transcription during the immune response (see e.g., Rao (1994) Immunology Today. 15:274-281; Crabtree et al. (1994) Annu. Rev. Biochem. 63:1045-1083). The involvement of NFAT is well established for the IL-2, IL-4, GM-CSF, and TNF-xcex1 cytokine genes in T cells. There is good but less extensive evidence for NFAT regulation of the IL-3, IL-5, IL-8, interferon-xcex3 (IFN-xcex3) and CD40L genes in T cells, the TNF-xcex1 gene in B cells, and the IL-4 and IL-5 genes in mast cells. Cooperative binding of NFAT has also been noted in the promoter regions of the IL-2, IL-4, IL-5, and CD40L genes, and in the GM-CSF enhancer.
Although NFATp MRNA has been detected in brain, heart, and skeletal muscle, NFATp protein expression has not been detected in bulk extracts of these tissues (see e.g., Wang et al. (1995) Annu. N.Y. Acad. Sci. 766:182-194). Consistent findings are that NFATp and NFATc mRNAs are expressed in peripheral lymphoid tissue (spleen, PBL), and that NFAT4/x MRNA is expressed at high levels in the thymus, suggesting a role in T cell development. NFAT3 mRNA is expressed at low levels in lymphoid tissues (see e.g., Hoey et al. (1995) Immunity 2:461-472), and thus this protein may be preferentially expressed outside the immune system.
The function of NFATp in the immune response has been explored by targeted disruption of the NFATp gene (see e.g., Hodge et al. (1996) Immunity 4:397-405; Xanthoudakis et al. (1996) Science 272:892-895). In both cases the targeted exon was in the DNA-binding domain, and its disruption resulted either in the expression of a deleted version of the protein without DNA-binding activity (see e.g., Hodge et al. (1996) Immunity 4:397-405), or in no protein expression (see e.g., Xanthoudakis et al. (1996) Science 272:892-895). Mice deficient in the NFAT gene developed normally, however, displayed splenomegaly with hyperproliferation of both B and T cells. They also displayed early defects in the transcription of multiple genes encoding cytokines and cell surface receptors, and a striking defect in in vivo IL-4 production. Despite this early defect in IL-4 production, certain immune responses were enhanced at later time points, particularly the development of Th2 cells, evidenced by increased IL-4 production and IgE levels (see e.g., Hodge et al. (1996) Immunity 4:397-405).
This invention pertains to methods and compositions relating to modulation of cartilage cell growth and/or differentiation by modulation of NFATp activity. It has now been discovered that NFATp plays a critical role in regulating the growth and/or differentiation of cartilage. The invention is based, at least in part, on the observation that mice lacking NFATp, as they age, develop tumors that arise from articular cartilage and from the surrounding extraarticular connective tissue. Cartilage cell lines established from these tumors were aneuploid and displayed loss of contact inhibition. These data demonstrate that NFATp represses cartilage cell division, extinguishes the cartilage phenotype and displays the properties of a tumor suppressor gene. To our knowledge this is the first demonstration of a role for NFATp in regulating cartilage cell growth and differentiation and the first example of a regulatory factor that affects the growth of cartilage cells in the upper proliferative zone of articular cartilage and cartilage cell differentiation at the level of the adult mesenchymal progenitor cell. Furthermore, the absence of NFATp results in induction of chondrogenesis in extraarticular connective tissue.
Accordingly, the invention provides methods for identifying compounds that modulate cartilage growth and/or differentiation, methods for modulating cartilage cell growth and/or differentiation using agents that modulate NFATp activity (e.g., methods to expand cartilage cells in culture by inhibiting NFATp activity in the cells such that proliferation of the cartilage cells is stimulated) and methods for diagnosing disorders associated with aberrant cartilage growth and/or differentiation (e.g., chondrosarcomas) based on assessing a change in the expression of NFATp (e.g., the level of expression or the form of NFATp expressed).
In one aspect, the invention pertains to methods of identifying a compound that modulates cartilage growth and/or differentiation by contacting cartilage cells deficient in NFATp with a test compound and determining the effect of the test compound on the growth and/or differentiation of the cartilage cells. The test compound is identified as a modulator of cartilage growth and/or differentiation based on the ability of the test compound to modulate the growth and/or differentiation of the cartilage cells deficient in NFATp. Cartilage cells deficient in NFATp can be contacted with a test compound in vivo in a non-human NFATp deficient animal, for example, a mouse, by administering the test compound to the non-human NFATp deficient animal. Cartilage cells deficient in NFATp may also be isolated from a non-human NFATp deficient animal and contacted with the test compound ex vivo by culturing the test compound with the isolated cartilage cells.
In another aspect, the invention pertains to a method of identifying a compound that modulates cartilage growth and/or differentiation using an indicator composition comprising NFATp protein, wherein a test compound that modulates the activity of NFATp is selected and then the effect of this selected compound on cartilage growth and/or differentiation is assessed. In the method, the indicator composition comprising NFATp first is contacted with each member of a library of test compounds. The test compound(s) that modulate the activity of NFATp protein are selected and the ability of the selected compound to modulate cartilage growth and/or differentiation is determined. The indicator composition can be, for example, a cell that expresses NFATp protein, a cell that has been engineered to express the NFATp protein by introducing an expression vector encoding the NFATp protein into the cell or a cell free composition. Alternatively, the indicator composition may be a cell or cell-free composition that includes an NFATp protein and a target molecule, and the ability of the test compound to modulate the interaction of the NFATp protein with a target molecule is monitored. In another embodiment, the indicator composition is an indicator cell which comprises an NFATp protein and a reporter gene responsive to the NFATp protein. The level of expression of the reporter gene can be used to determine the ability of a test compound to modulate the activity of NFATp protein by producing an indicator cell that contains a recombinant expression vector encoding the NFATp protein and a vector comprising an NFATp-responsive regulatory element operatively linked a reporter gene. The indicator cell is contacted with a test compound and the level of expression of the reporter gene in the indicator cell in the presence of the test compound is determined. By comparing the level of expression of the reporter gene in the indicator cell in the presence of the test compound with the level of expression of the reporter gene in the indicator cell in the absence of the test compound, a compound of interest that modulates the activity of NFATp protein can be determined.
In another aspect, the invention pertains to a method for modulating cartilage cell growth and/or differentiation by contacting cartilage cells with a modulator of NFATp activity such that the growth and/or differentiation of the cartilage cells is modulated. In one aspect, this modulatory method pertains to methods of expanding cartilage cells in vitro, through culture of the cells with an inhibitor of NFATp activity such that proliferation of the cartilage cells is stimulated. In another aspect, this modulatory method pertains to methods of modulating aberrant cartilage growth and/or differentiation in a subject by administering to the subject a therapeutically effective amount of a specific modulator of NFATp activity such that aberrant cartilage growth and/or differentiation in a subject is modulated. In one embodiment, the modulator inhibits NFATp activity, for example, an antisense oligonucleotide, an intracellular antibody or a peptide that inhibits the interaction of NFATp with calcineurin. In another embodiment, the modulator stimulates NFATp activity, for example, an expression vector encoding NFATp. The modulator can be administered directly to an articulation site of a subject, or can be contacted ex vivo with cartilage cells isolated from a subject, followed by administration of the cartilage cells back into the subject.
In another aspect, the invention pertains to a method of diagnosing a subject for a disorder associated with aberrant cartilage growth and/or differentiation by detecting a change in expression of NFATp in cartilage cells of a subject suspected of having a disorder associated with aberrant cartilage growth and/or differentiation. The expression of NFATp in cartilage cells of a subject suspected of having the disorder is compared to the expression of NFATp in cartilage cells of a control subject without the disorder. The diagnosis for a disorder in a subject is based on a change in expression of NFATp (e.g., the level or form of NFATp) in cartilage cells relative to a control subject. For example, elevated levels of NFATp expression or expression of a constitutively active mutant form of NFATp may be associated with a disorder characterized by deficient cartilage growth and/or differentiation, while reduced levels of NFATp expression or expression of an inactive mutant form of NFATp may be associated with a disorder characterized by increased cartilage cell growth and/or differentiation, e.g., chondrosarcomas.
Kits for performing the various methods of the invention are also encompassed by the invention.